Bioenergetic analysis of human peripheral blood mononuclear cells.

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.

Disruption of doubly uniparental inheritance of mitochondrial DNA in hybrid mussels (Mytilus edulis x M. galloprovincialis).

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA inheritance termed doubly uniparental inheritance (DUI). Females are homoplasmic for the F mitotype which is inherited maternally, whereas males are heteroplasmic for this and the paternally inherited M mitotype. In areas where species distributions overlap a varying degree of hybridization occurs; yet genetic differences between allopatric populations are maintained. Observations from natural populations and previous laboratory experiments suggest that DUI may be disrupted by hybridization, giving rise to heteroplasmic females and homoplasmic males. We carried out controlled laboratory crosses between Mytilus edulis and M. galloprovincialis to produce pure species and hybrid larvae of known parentage. DNA markers were used to follow the fate of the F and M mitotypes through larval development. Disruption of the mechanism which determines whether the M mitotype is retained or eliminated occurred in an estimated 38% of M. edulis x M. galloprovincialis hybrid larvae, a level double that previously observed in adult mussels from a natural M. edulis x M. galloprovincialis hybrid population. Furthermore, reciprocal hybrid crosses exhibited contrasting types of DUI disruption. The results indicate that disruption of DUI in hybrid mussels may be associated with increased mortality and hence could be a factor in the maintenance of genetic integrity for each species.

Spontaneous mutation spectra in supF: comparative analysis of mammalian cell line base substitution spectra.

The last decade has seen a dramatic accumulation of mutation data from reporter genes utilized in mutagenesis experiments involving DNA reactive agents allowing comparisons for the mutagenic potential between many different mutagens. When analysing chemically induced mutation spectra it is important to establish the potential spontaneous background before drawing conclusions concerning specific chemically induced hotspots. A major mutation reporter system gene used in mammalian cells is the supF suppressor tRNA gene. The Mammalian Gene Mutation Database (MGMD) contains a considerable number of supF spontaneous mutations permitting a thorough analysis of spontaneous mutations in mammalian cell lines from different species and tissues. Analyses of spontaneous mutation spectra were performed using a range of statistical techniques. Spontaneous mutations were observed at 82.4% of the nucleotides in the supF suppressor tRNA sequence although the pattern of significant hotspots differed between cell lines. Our analyses of spontaneous mutation spectra show considerable variation both within and between cell lines for the distributions of spontaneous mutations occurring with no clear tissue or species-specific patterns emerging. In addition, spectra derived from supF recovered from liver and skin of transgenic mice, were similar to each other, but showed significant differences from many in vitro spectra. The most common base substitutions were G:C>TA transversions and G:C>A:T transitions, although levels of each type differed between cell lines. There was also variation between cell lines for the most mutable dinucleotides, however, significant hotspots were frequently observed at CpG sites and sequences containing GG/CC. We conclude that the number of varying distributions and potential hotspots for spontaneous mutations should thus be considered when comparing chemically induced mutation spectra in supF. The spectra presented here will be a useful reference for analysis and re-analysis of chemically induced spectra as well as for use in comparison with the spontaneous spectra of other gene systems.

Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour.

Warthin's tumour of the salivary glands is composed of oncocytic cells containing excessive numbers of mitochondria which show frequent structural abnormalities and reduced metabolic function. Recent evidence of a strong association between cigarette smoking and the occurrence of Warthin's tumour prompted this study, to look for evidence of damage to mitochondrial DNA (mtDNA) that could be the result of an increase in oxidative stress; two-colour fluorescence in situ hybridization (FISH) was developed to show the distribution of mitochondria with deleted mtDNA in paraffin wax-embedded material. Approximately 10% of mtDNA bears the 'common' 4977 bp deletion. Using the polymerase chain reaction (PCR), the 4977 bp deletion was further quantified, in Warthin's tumour and age-matched normal parotid control tissue. Whilst the deletion was present in all parotid tissue, its presence was significantly higher in oncocytic tumour cells. In a small number of controls, there was a trend towards higher concentrations of the deletion in smokers.

Microsatellite marker based genetic linkage maps of Oreochromis aureus and O. niloticus (Cichlidae): extensive linkage group segment homologies revealed.

Partial genetic linkage maps, based on microsatellite markers, were constructed for two tilapia species, Oreochromis aureus and Oreochromis niloticus using an interspecific backcross population. The linkage map for O. aureus comprised 28 markers on 10 linkage groups and covered 212.8 CM. Nine markers were mapped to four linkage groups on an O. niloticus female linkage map covering 40.6 CM. Results revealed a high degree of conservation of synteny between the linkage groups defined in O. aureus and the previously published genetic linkage map of O. niloticus.

Interspecies transfer of female mitochondrial DNA is coupled with role-reversals and departure from neutrality in the mussel Mytilus trossulus.

Mussels of the genus Mytilus have distinct and highly diverged male and female mitochondrial DNA (mtDNA) genomes with separate routes of inheritance. Previous studies of European populations of Mytilus trossulus demonstrated that 33% of males are heteroplasmic for a second mtDNA genome of increased length and that hybridization with Mytilus edulis does not block mtDNA introgression, in contrast to reports for American populations. Here, we demonstrate that the female mtDNA type of M. edulis has replaced the resident female mtDNA type of European M. trossulus. This is supported by COIII sequence data indicating that the female mtDNA of European M. trossulus is very similar to that of M. edulis and that in phylogenetic trees, the mtDNAs of these two species cluster together but separately from American M. trossulus sequences, the latter not being disturbed by introgressive hybridization. We also provide evidence that the mtDNA genome of increased length found in heteroplasmic males of European M. trossulus derives from a recent partition of an introgressed M. edulis female type into the male route of transmission. Neutrality tests reveal that European populations of M. trossulus display an excess of replacement polymorphism within the female mtDNA type with respect to conspecific American populations, as well as a significant excess of rare variants, of a similar magnitude to those previously reported for the invading European M. edulis mtDNA. Results are consistent with a nearly neutral model of molecular evolution and suggest that selection acting on European M. trossulus mtDNA is largely independent of the nuclear genetic background.

Nonneutral evolution and differential mutation rate of gender-associated mitochondrial DNA lineages in the marine mussel Mytilus.

Mussels have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally, and the F type is transmitted maternally. To test hypotheses of the molecular evolution of both mtDNA genomes, 50 nucleotide sequences were obtained for 396 bp of the COIII gene of European populations of Mytilus edulis and the Atlantic and Mediterranean forms of M. galloprovincialis. Analysis based on the proportion of synonymous and nonsynonymous substitutions indicate that mtDNA is evolving in a non-neutral and complex fashion. Previous studies on American mussels demonstrated that the F genome experiences a higher purifying selection and that the M genome evolves faster. Here we show that these patterns also hold in European populations. However, in contrast to American populations, where an excess of replacement substitution between F and M lineages has been reported, a significant excess of replacement polymorphism within mtDNA lineages is observed in European populations of M. galloprovincialis. European populations also show an excess of replacement polymorphism within the F but not within the M genome with respect to American M. trossulus, as well as a consistent pattern of excess of rare variants in both F and M genomes. These results are consistent with a nearly neutral model of molecular evolution and a recent relaxation of selective constraints on European mtDNA. Levels of diversity are significantly higher for the M than F genome, and the M genome also accumulates synonymous and nonsynonymous substitutions at a higher rate, in contrast with earlier reports where no difference for the synonymous rate was observed. It is suggested that a subtle balance between relaxed selection and a higher mutation rate explains the faster evolutionary rate of the M lineage.

Sex-biased heteroplasmy and mitochondrial DNA inheritance in the mussel Mytilus galloprovincialis Lmk.

An exceptional mode of mtDNA inheritance involving separate maternal and paternal transmission routes has been reported recently in the mussel Mytilus edulis. This mode of inheritance provides an explanation for the high levels of heteroplasmy for two highly diverged genomes observed in males of this species. Here we provide evidence for a similar pattern of heteroplasmy in Atlantic and Mediterranean forms of the related mussel M. galloprovincialis. The results support the hypothesis that this mode of mtDNA inheritance has an ancient origin. In addition, the detection of some heteroplasmic females suggests preferential, rather than exclusive, transmission within male and female lines of descent. We also present evidence that the two highly diverged genomes display a parallel split between the Atlantic and Mediterranean forms, consistent with neutral evolution.

Multilocus DNA fingerprinting and RAPD reveal similar genetic relationships between strains of Oreochromis niloticus (Pisces: Cichlidae).

Two molecular techniques which reveal highly variable DNA polymorphisms, RAPD and multilocus DNA fingerprinting, were used to evaluate genetic diversity between six aquacultural strains of Oreochromis niloticus (tilapia) from the Philippines. The results using both techniques were in close agreement. Within-strain heterozygosity values were similar and were correlated between the two data sets, but statistical errors associated with the RAPD data set were lower. Although genetic distances between strains were greater using DNA fingerprinting, the distances measured using both methods were significantly correlated. Both methods were useful in estimating variation between strains, but they offered different advantages. RAPD was technically easier to perform and produced results with low statistical error, whereas DNA fingerprinting detected greater genetic differentiation between strains. The theoretical basis for using RAPD and multilocus minisatellite markers for population studies is discussed.

Sex-limited mitochondrial DNA transmission in the marine mussel Mytilus edulis.

Mitochondrial DNA (mtDNA) was thought to be inherited maternally in animals, although paternal leakage has been reported in mice and Drosophila. Recently, direct evidence of extensive paternal inheritance of mtDNA has been found in the marine mussel Mytilus. We give evidence that whereas female mussels are homoplasmic for a genome that is transmitted to eggs, male mussels are heteroplasmic for this genome and for a second genome that is transmitted preferentially to sperm. The results provide support for the existence of separate male and female routes of mtDNA inheritance in mussels. The two genomes show a base sequence divergence exceeding 20% at three protein coding genes, consistent with long term maintenance of the heteroplasmic state. We propose that the two genomes differ in fitness in males and females, possibly as a result of interaction with nuclear genes.

Application of the RAPD technique in tilapia fish: species and subspecies identification.

Random Amplified Polymorphic DNA (RAPD) analysis was applied to three species of the tilapia genus Oreochromis and four subspecies of O. niloticus. Thirteen random 10-mer primers were used to assay polymorphisms within and between populations. Different RAPD fragment patterns were observed for different species, although not always for different subspecies. Evidence is presented that RAPD markers might be useful for systemic investigation at the level of species and subspecies.

A quantitative test of the neutral theory using pooled allozyme data.

Neutral theory predicts a positive correlation between the amount of polymorphism within species and evolutionary rate. Previous tests of this prediction using both allozyme and DNA data have led to conflicting conclusions about the influence of selection and mutation drift. It is argued here that quantitative conclusions about the adequacy of neutral theory can be obtained by analyzing genetic data pooled from many sources. Using this approach, a large database containing information on allozyme variation in over 1500 species is used to examine the relationship between heterozygosity and genetic distance. The results provide support for the hypothesis that a major percentage of protein variation can be explained by variation in neutral mutation rate, and a minor percentage by strong selection.

A study of interlocus allozyme heterozygosity correlations: implications for neutral theory.

Using a database of allozyme studies, correlations in heterozygosity between selected enzyme loci (MDH, alpha GPDH, IDH, 6PGDH, LDH, SOD, AAT, PGM, EST, PGI) were calculated across vertebrate species. Large and positive correlations were observed with untransformed heterozygosity values. However, after transformation to correct for mean species heterozygosity, correlations were substantially reduced and median values were closer to zero. Some enzymes were more often involved in significant correlations than others, and correlations calculated across species within vertebrate classes were significant for different enzyme pairs in different classes. There was no evidence that significant correlations occurred primarily between functionally related enzymes. It is suggested that the observed correlations are best explained by variation between enzyme loci in functional constraint and effective neutral mutation rate.

Sex determination in the genus Oreochromis : 1. Sex reversal, gynogenesis and triploidy in O. niloticus (L.).

Established techniques of genetic manipulation were used to elucidate sex-determining mechanisms in the commercially important tilapia, Oreochromis niloticus. Analysis of sex ratios from single-pair matings of normal broodstock showed these to be heterogeneous, with an asymmetrical frequency distribution. Data were homogeneous, with the exclusion of a number of broods with sex ratios not significantly different from 3∶1 (male: female), and further progeny testing revealed atypical female heterogamety in the parents of these broods. Analysis of sex ratios from complete diallele-type crosses using five males and five females demonstrated no association between male parent, female parent and progeny sex ratio. Sex ratios of gynogens (0∶1) and triploids (1∶1), and from progeny testing of sex-reversed males (0∶1) and sex-reversed females (3∶1), provide evidence for female homogamety in this species. Progeny testing of male gynogens derived from sex-reversed females demonstrated recombination between the centromere and the sex-determining locus (68.9%). Novel YY "supermales" were shown to be viable and produced all-male offspring. It was concluded that this species exhibits monofactorial, genotypic sex determination with male heterogamety. However, rare autosomal or environmental sex-modifying factors may account for occasional deviations from expected sex ratios.

Sex determination in the genus Oreochromis : 2. Sex reversal, hybridisation, gynogenesis and triploidy in O. aureus Steindachner.

Sex ratios from 62 single-pair matings of normal broodstock O. aureus were highly heterogeneous with an overall deficit of males (41.4%). Peaks in the sex ratio frequency distribution occurred at 1∶1, 3∶5 and 1∶3 (male∶female). Hybridisation of O. aureus with O. mossambicus, O. spilums and O. niloticus produced highly variable sex ratios, suggesting a complexity of hybrid sex determination. Few valid inferences could be made regarding intraspecific sex determination from these hybrid data. Sex ratios from progeny testing of sex-reversed males (1∶3) and most sex-reversed females (1∶0) provide evidence for female heterogamety in O. aureus. Several aberrant ratios observed suggest Mendelian inheritance of an autosomal recessive gene (F,f), epistatic to the major sex-determining gene (W,Z). Sex ratios of triploids and gynogens support the hypothesis of recombination between the centromere and the major sex-determining locus. Progeny testing of a female mitogyne demonstrated the viability of a novel WW "superfemale", which gave only female offspring. Not all data could be explained by a two-factor model of sex determination. Further exceptional sex ratios may be accounted for by rare autosomal or environmental sex-modifying factors. It is concluded that O. aureus has a multifactorial mechanism of sex determination with the underlying primary mechanism of female heterogamety.

Restriction site mutation analysis, a proposed methodology for the detection and study of DNA base changes following mutagen exposure.

We propose a methodology for the detection of DNA base changes in restriction enzyme recognition sites. The restriction site mutation (RSM) technique is based upon the detection of DNA sequences resistant to the cutting action of specific restriction enzymes and the amplification of these resistant sequences using the polymerase chain reaction. As outlined, the RSM method has the potential for use in the study of induced base changes in any species, tissue and genes of known DNA sequence for which unique DNA primers are available, and which contains a number of unique restriction enzyme recognition sites.

Directional selection in lines founded from different parts of the phenotypic distribution of sternopleural chaetae number in Drosophila melanogaster.

Divergent directional selection lines were initiated from base populations founded from parents taken from different parts of the sternopleural chaetae distribution in a cage population of Drosophila melanogaster. Lines founded from parents taken from the central part of the distribution showed greater response and higher realised heritability than lines derived from parents with extreme high or extreme low chaetae number. The results suggest that centrally derived phenotypes have higher heterozygosity for chaetae factors than extreme phenotypes and that these factors have a large effect on the character.